小麦TaHMA2转运蛋白的底物特异性和调节籽粒Zn转运和累积的机制研究

Zinc is an essential element for human, Millions of people worldwide are suffering from nutritional deficiency of zinc, the low zinc content of staple crops like wheat (Triticum aestivum) is the major cause in developing country. Enhancement of Zn translocation and partitioning from root to shoot is one of the strategies in biofortification. Heavy metal ATPase 2 (HMA2) plays important roles in regulation Zn2+ uptake, transport and accumulation, and it could be candidate genes for engineering modification of zinc concentration partitioning in grains. However, the molecular mechnism of Zn accumuated in wheat grains remains fragmentary. To enhance the Zn concentration in grains of wheat, based on our previous experimental results, we will focus on the substrate specificity and activity rate of various mutant version of TaHMA2 transporting protein by the truncation of the C-terminal sequence and a single base-pair mutation, the level and cell and tissue specificity of TaHMA2 promoter activity using the 5' end deletion analysis, examining how TaHMA2 alters the distribution of Zn within the leaf and grain by imaging with the fluorescent Zn indicator Zinpyr-1 and synchrotron radiation X-ray fluorescence (SRXRF). The relationship between the expression pattern of TaHMA2 and content of Zn and other essential metals (Mn, Fe, Cu) in grain was investigated. Finally, we could figure out the mechanism of TaHMA2 modification the Zn translocation and distribution in grain. The results will provide theoretic knowledge for zinc biofortification of staple crops.

锌作为人体必需的微量元素，粮食作物中锌含量偏低是导致许多发展中国家人体锌缺乏的重要原因，提高作物锌向地上部的转运和分配是解决人类锌营养匮乏的重要途径之一。重金属转运蛋白2（HMA2）在锌的吸收、转运、分配和积累等各个过程起着非常重要的作用，是调控籽粒Zn累积的一个候选基因，然而，小麦籽粒Zn累积的机制未见报道。为了强化小麦籽粒的Zn营养，本项目拟在前期工作基础上，利用C-末端缺失和单碱基突变技术研究小麦TaHMA2突变体的底物专一性和转运活性，用5'末端缺失方法分析TaHMA2启动子的活性和表达的组织部位特异性，用Zn荧光探针和用微区同步辐射技术（SRXRF）分析TaHMA2如何调控小麦叶片和籽粒中Zn的分配，揭示TaHMA2基因的表达与Zn和其它必需元素含量的关系，最终勾画出TaHMA2调控籽粒Zn移位和分配的分子机制。为作物的锌营养强化提供理论基础。

土壤肥力的退化和环境污染导致耕地出现缺锌(Zn)和镉(Cd)积累情况，严重影响作物生长发育。因此增加作物的必需元素，减少有害重金属的累积是农业生产中一个紧迫的环境问题。通过分子育种的方法是潜在解决作物缺锌和镉毒害问题的途径。小麦重金属ATP酶2(TaHMA2)在Zn2+/Cd2+累积和转运中起重要作用。本研究我们报道了过表达TaHMA2的酵母能累积更多的Cd2+，对Cd2+非常敏感。而过表达TaHMA2拟南芥能提高对Zn2+/Cd2+的耐受性，且增加对Zn2+/Cd2+从地下部到地上部的转运能力。TaHMA2在酵母和拟南芥中均定位在细胞质膜上。研究还发现TaHMA2的N端金属结合域 (N-MBD)上包含CCxxE和CPC基序，两个基序上的半胱氨酸（Cys）突变后，过表达酵母和拟南芥在Zn2+/Cd2+处理下丧失了对Zn2+/Cd2+的耐受性和转运能力，而谷氨酸（Glu）的突变不影响对Zn2+/Cd2+的转运。同时C末端截断的TaHMA2过表达酵母和拟南芥降低对Zn2+/Cd2+的耐受性和转运，结果表明N-MBD上CCxxE和CPC基序中半胱氨酸对Zn2+/Cd2+的结合和转运起至关重要的作用。本课题研究成果为小麦TaHMA 2基因在作物分子设计育种中的应用及其调节作物中金属元素的离子平衡提供了理论依据。

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